A type of anticancer agent that has attracted intense current interest is an antibody-drug conjugate (ADC), also known as an immunoconjugate. In an ADC, a therapeutic agent (drug) is covalently linked to an antibody whose antigen is expressed by a cancer cell. The antibody, through its binding to the antigen, serves to deliver the ADC to the cancer. Once there, cleavage of the covalent link or degradation of the antibody results in the release of the therapeutic agent at the cancer site. Conversely, while the ADC is circulating in the blood system, the therapeutic agent is held inactive because of its covalent linkage to the antibody. Thus, the therapeutic agent in an ADC can be much more potent (i.e., cytotoxic) than ordinary chemotherapy agents because of its localized release. Thus, an ADC comprises three components: (1) the antibody, (2) a drug, and (3) a linker covalently joining the antibody and the drug. For a review on ADCs, see Schrama et al. 2006.
A key step in the preparation of an ADC is the covalent joining step, also referred to as the conjugation step. Many methods having been disclosed for effecting conjugation. One that has engendered substantial recent interest is conjugation mediated by transglutaminase (TGase), in particular bacterial transglutaminase (BTG). See, for example, Jeger et al. 2010.
BTG forms an amide bond between the carboxamide side chain of a glutamine (the amine acceptor) and the ε-amino group of a lysine (the amine donor). Specificity-wise, transglutaminase is selective regarding the glutamine residue, e.g. requiring that it be located in a flexible part of a protein loop and flanked by particular amino acids, but is promiscuous regarding the lysine residue, for example readily accepting the amino group of an alkyleneamino compound as a lysine ε-amino surrogate. See Fontana et al. 2008.
In a typical BTG-mediated conjugation the glutamine residue is located on the antibody, while the amino group is located on the linker-drug moiety, as shown below:
In this scheme, the antibody acts as an amine acceptor and the H2N-[Linker]-[Drug] moiety acts as an amine donor.
The location of a glutamine residue on a polypeptide chain has a large effect on its susceptibility to BTG mediated transamidation. None of the glutamine residues on an antibody are normally BTG substrates and some modification of the antibody is necessary to induce BTG susceptibility. Typically, an antibody is glycosylated at asparagine 297 (N297) of the heavy chain (N-linked glycosylation). Jeger et al. 2010 discovered that deglycosylation of the antibody, either by eliminating the glycosylation site through an N297A substitution or post-translation enzymatic deglycosylation, renders nearby glutamine 295 (Q295) BTG-susceptible. They also showed that an N297Q substitution not only eliminates glycosylation, but also introduces a second glutamine residue (at position 297) that too is an amine acceptor. Thus, simple deglycosylation generates two BTG-reactive glutamine residues per antibody (one per heavy chain, at Q295), while an antibody with an N297Q substitution will have four BTG-reactive glutamine residues (two per heavy chain, at positions Q295 and Q297).
Disclosures relating to the transglutaminase-mediated preparation of ADCs include: Dennler et al. 2014, Innate Pharma 2013, Jeger et al. 2010, Pons et al. 2013, and Strop et al., 2013.
In particular, Pons et al. 2013 disclose the modification of an antibody with a four-amino acid glutamine-containing tag, which can be located at the C-terminus of one of its chains, to make it transglutaminase-reactive. (Attachment of tags or extensions to the C-terminus of an antibody chain in other contexts has also been disclosed. See, e.g., Liu et al. 2014.)
Other transglutaminase disclosures, more generally relating to the labeling or modification of proteins (including antibodies), include: Bregeon 2014, Bregeon et al. 2013 and 2014, Chen et al. 2005, Fischer et al. 2014, Kamiya et al. 2011, Lin et al. 2006, Mero et al. 2009, Mindt et al. 2008, Sato 2002, Sato et al. 2001, and Sugimura et al. 2007.
Full citations for the documents cited herein by first author or inventor and year are listed at the end of this specification.